Preparation and evaluation method of animal model of phlegm-fire disturbing spirit mania

Abstract

The application discloses a preparation and evaluation method of an animal model of phlegm-fire disturbing spirit mania, and belongs to the technical field of animal models.The application aims to solve the technical problems of the lack of TCM syndrome properties and the single evaluation system of the existing mania model; the method takes C57BL / 6J mice as objects, uses high-fat feed combined with sucrose drinking water to feed for four weeks to shape phlegm-dampness and heat constitution, and then uses GBR12909 intraperitoneal injection for 14 days to induce mania behavior, and the total cycle is six weeks. Model evaluation integrates four types of indexes of ethology, TCM syndrome, metabolism and inflammation-neurotransmitter, and realizes multi-dimensional comprehensive evaluation. The method is simple in operation, stable in model formation, and can provide a practical experimental tool for the standardized evaluation of the phlegm-fire disturbing spirit mania model and the research and development of related drugs.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically to a method for preparing and evaluating an animal model of manic-depressive disorder caused by phlegm-fire disturbance. Background Technology

[0002] Mania is a form of mood disorder characterized by flight of ideas, elevated mood, and increased speech and activity. Most patients have a tendency for recurrent episodes, and some may experience residual symptoms or develop chronic mania. The etiology of mania is not fully understood, but research suggests a close relationship with genetic, biological, and psychological factors. Treatment primarily involves antipsychotic drugs, but these drugs generally have limitations such as a narrow safe dosage window, significant adverse reactions, and low patient compliance. Traditional Chinese medicine (TCM) has shown certain advantages in the treatment of mania; however, due to the unclear pathogenesis, further rigorous research is needed to investigate the intrinsic relationship between central neurotransmitters and TCM responses based on TCM syndrome differentiation and treatment, thereby ensuring the rationality and superiority of TCM in treating mania. Therefore, animal models have become an important means of revealing its mechanisms and intervention strategies.

[0003] Currently, various animal models of mania have been established both domestically and internationally, mainly including acute amphetamine or cocaine injection models, sodium iodate models, and GBR12909 monotherapy models. However, existing models still have the following shortcomings: First, the model construction is mainly based on single Western medical neurobiological mechanisms, lacking guidance from traditional Chinese medicine (TCM) theory, making it difficult to reflect the TCM syndrome attributes, and therefore unsuitable as animal models for evaluating the efficacy of TCM; second, they fail to simulate the constitutional basis of manic episodes, resulting in an incomplete pathological connotation of the models; third, there is a general lack of systematic and objective TCM syndrome evaluation standards, making it difficult to incorporate TCM pathogenesis-related indicators into the model evaluation methods, thus restricting the scientific rigor and objectivity of TCM efficacy evaluation.

[0004] In summary, there is an urgent need to establish a novel animal model of mania that can not only reflect the TCM pathogenesis of "phlegm-fire disturbing the mind" in mania, but also has a modern neurobiological basis and is equipped with a standardized TCM syndrome evaluation method. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing and evaluating an animal model of manic-depressive disorder caused by phlegm and fire disturbing the mind.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] On the one hand, this application provides a method for preparing an animal model of manic-depressive disorder caused by phlegm-heat. The modeling method is as follows: Four-week-old male C57BL / 6J mice are selected and fed a high-fat diet and 20% sucrose in drinking water for four weeks to simulate the constitution of phlegm-dampness-heat accumulation. Starting from the fifth week, GBR12909 is injected intraperitoneally at the same time every day for 14 consecutive days to induce manic-like behavior.

[0010] Specifically, the high-fat diet has a fat energy ratio greater than or equal to 60%.

[0011] Specifically, the GBR12909 is prepared into a 1 mg / mL working solution using a solvent composed of 2% DMSO, 40% PEG300, 5% Tween-80 and 53% physiological saline, and the intraperitoneal injection dose is 10 mg / kg.

[0012] Specifically, the same time point is intraperitoneal injection between 8:00 and 10:00 every day to ensure the consistency of the diurnal rhythm.

[0013] On the other hand, this application provides an evaluation method for mouse models obtained by the above-mentioned modeling method, including the following steps:

[0014] Step 1: Conduct behavioral evaluations on the model mice, including the central open field test, sucrose preference test, forced swimming test, three-compartment social test, rotarod test, elevated cross maze test, and water maze test, to determine the degree of manic-like core behavioral abnormalities in the animals.

[0015] Specifically, the total distance the mice under evaluation moved in the central open field experiment was used as the sole scoring indicator for the behavioral dimension. The mean total distance of the normal control group mice in the same evaluation batch was used as the baseline value and set to 0 points, while the maximum observed total distance of the model group mice in the same evaluation batch was used as the upper limit value and set to 100 points. The behavioral dimension score of the mice under evaluation was calculated according to the following formula:

[0016]

[0017] When the calculated behavioral dimension score is less than 0, it is recorded as 0; when it is greater than 100, it is recorded as 100. When the upper limit is less than or equal to the baseline value, the behavioral dimension score of the mice in this batch of evaluations is recorded as 0. Other behavioral indicators are only used to assist in judging the behavioral characteristics of the model and to exclude interfering factors such as depressive-like behavior, motor dysfunction, anxiety-like behavioral abnormalities, and cognitive impairment, and are not included in the behavioral dimension score.

[0018] Step 2: Evaluate the TCM syndrome of phlegm-fire disturbing the mind in the modeled mice, including changes in hair luster, oral and nasal mucosa color, fecal characteristics and water intake, and incorporate them into the TCM phlegm-fire disturbing the mind syndrome dimension calculation.

[0019] Specifically, in step two, various indicators of TCM syndromes were obtained in the mice, such as hair luster, color of oral and nasal mucosa, fecal characteristics, and changes in water intake. The original scores were calculated using a grading scoring method and converted to a score of 0-100. The grading scoring method is shown in Table 1.

[0020] Table 1. Traditional Chinese Medicine Syndrome Grading and Scoring Table

[0021]

[0022] The raw scores of the four indicators are added together to obtain the total raw score of the syndrome, and the score of the TCM phlegm-fire disturbing the mind syndrome dimension is calculated according to the following formula:

[0023]

[0024] Step 3: Perform metabolic evaluation on the model mice, including weight gain rate, fasting blood glucose, triglycerides, total cholesterol and low-density lipoprotein cholesterol, and incorporate them into the metabolic dimension calculation.

[0025] Specifically, the mouse weight gain rate in step three is calculated using the following formula:

[0026]

[0027] Specifically, in step three, the mean values ​​of the corresponding indicators (except for high-density lipoprotein cholesterol) of the normal control group mice in the same evaluation batch were used as the metabolic baseline values, and the maximum observed values ​​of the corresponding indicators of the model group mice in the same evaluation batch were used as the upper limits of metabolism. The scores of the corresponding individual indicators were calculated according to the following formula:

[0028]

[0029] For high-density lipoprotein cholesterol (HDL-C), the mean HDL-C value of normal control mice in the same evaluation batch was used as the baseline, and the minimum observed HDL-C value of model mice in the same evaluation batch was used as the lower limit, calculated according to the following formula:

[0030]

[0031] When any single indicator score is less than 0, it is recorded as 0 points; when it is greater than 100 points, it is recorded as 100 points. When the upper limit of the elevated indicator is less than or equal to the corresponding benchmark value, or the lower limit of high-density lipoprotein cholesterol is greater than or equal to the corresponding benchmark value, the corresponding single indicator score is recorded as 0 points.

[0032] The scores for metabolic indicators are calculated using the following formula:

[0033]

[0034] Step 4: Evaluate the inflammation-neurotransmitter levels of the model mice, including serum IL-6, IL-1β, TNF-α, striatal dopamine levels, and the prefrontal cortex glutamate / γ-aminobutyric acid ratio, and include them in the calculation of the inflammation-neurotransmitter dimension.

[0035] Specifically, in step four, the mice used the above indicators from the normal control group mice in the same evaluation batch as the baseline values ​​for the corresponding indicators, and the maximum observed values ​​of the corresponding indicators from the model group mice in the same evaluation batch as the upper limits for the corresponding indicators. The scores for each individual indicator were calculated according to the following formula:

[0036]

[0037] When the calculation result is less than 0 points, it is recorded as 0 points; when it is greater than 100 points, it is recorded as 100 points. When the upper limit value is less than or equal to the corresponding benchmark value, the corresponding single indicator score is recorded as 0 points.

[0038] The inflammation-neurotransmitter dimension score is calculated according to the following formula:

[0039]

[0040] Step 5: Calculate a comprehensive score based on the results obtained in steps 1-4 to evaluate the construction of the mouse model; when the comprehensive score of the model is greater than or equal to 60 points and the behavioral dimension score is greater than or equal to 60 points, the animal model of phlegm-fire disturbing the mind and mania is determined to be successfully constructed.

[0041] Specifically, the comprehensive scoring formula is: (TCM phlegm-fire disturbing the mind syndrome score × 15%) + (metabolic index score × 25%) + (inflammation-neurotransmitter score × 35%) + (behavioral dimension score × 25%).

[0042] (III) Beneficial Effects

[0043] This invention is based on the classic TCM pathogenesis theory of "emotional distress leading to phlegm and saliva production, and phlegm-fire disturbing the mind," combined with clinical manifestations of mania such as elevated mood, irritability, and increased activity. It establishes a phlegm-dampness-heat constitution using a high-fat diet and sucrose-containing water intake, and combines this with chronic intervention using the dopamine transporter inhibitor GBR12909 to induce hyperfunction of the central dopamine system, thereby constructing an animal model of mania with TCM syndrome characteristics of phlegm-fire disturbing the mind. In terms of evaluation strategy, it pioneers a four-dimensional weighted scoring system based on TCM syndrome, metabolism, inflammation-neurotransmitter, and behavior, achieving a comprehensive, objective, and standardized assessment of model quality. This invention achieves an organic unity between TCM syndrome theory and modern neuroscience methods, providing a valuable experimental tool for basic research and new drug development in mania caused by phlegm-fire disturbing the mind. Attached Figure Description

[0044] Figure 1 The scoring results and evaluation criteria for the TCM syndrome of phlegm-fire disturbing the mind; compared with the control group, * P < 0.05, ** P < 0.01. Compared with the GBR12909 group, # P < 0.05, ## P<0.05.

[0045] Figure 2 The results show the behavioral scores for sucrose preference in each group; compared with the control group, ns indicates no statistical significance; compared with the GBR12909 group, ns indicates no statistical significance.

[0046] Figure 3 The results of the central open field behavioral scores for each group; compared with the control group, *** P<0.001; compared with the GBR12909 group, # P < 0.05, ### P < 0.001.

[0047] Figure 4 The forced swimming behavior scores for each group; compared with the control group, *** P < 0.001; ns indicates no statistical significance compared with the GBR12909 group.

[0048] Figure 5 The results of the three-room social behavior assessment for each group; compared with the control group, *** P < 0.001, ns indicates no statistical significance; compared with the GBR12909 group, ## P < 0.01, ### P<0.001.

[0049] Figure 6The results of bar rotation behavior scores for each group are shown below. Compared with the control group, ns indicates no statistical significance. Compared with the GBR12909 group, ns indicates no statistical significance.

[0050] Figure 7 The behavioral scores for the elevated cross maze are shown for each group; compared with the control group, ns indicates no statistical significance; compared with the GBR12909 group, ns indicates no statistical significance.

[0051] Figure 8 The results show the behavioral scores for the water maze in each group; compared with the control group, ns indicates no statistically significant difference; compared with the GBR12909 group, # P < 0.05, ns indicates no statistical significance.

[0052] Figure 9 The results of weight gain rate, fasting blood glucose, and blood lipid-related indicators for each group were compared with those of the control group. *** P < 0.001, ns indicates no statistical significance; compared with the GBR12909 group, ### P < 0.001, ns indicates no statistical significance.

[0053] Figure 10 The results of serum inflammatory factor indicators in each group; compared with the control group, * P < 0.05, *** P < 0.001, ns indicates no statistical significance; compared with the GBR12909 group, ### P < 0.001.

[0054] Figure 11 The results of neurotransmitter-related indicators for each group; compared with the control group, * P < 0.05, ** P<0.01, *** P < 0.001, ns indicates no statistical significance; compared with the GBR12909 group, ### P < 0.001. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0056] Example 1

[0057] Construction of an animal model of mania based on the syndrome of phlegm-fire disturbing the mind

[0058] 1.1 Laboratory Animals

[0059] Four-week-old male C57BL / 6J mice, SPF grade, weighing 14±2 g, were purchased from Beijing Spefolk Biotechnology Co., Ltd. All experimental procedures were approved by the Animal Ethics Committee of Henan University of Traditional Chinese Medicine, approval number DWLL202205008. Husbandry conditions: All animals were housed in the Animal Experiment Center of Henan University of Traditional Chinese Medicine, three per cage, with free access to food and water, a 12-hour diurnal cycle (lights on at 8:00 AM), a room temperature of 22.6±0.4℃, and a relative humidity of 64.2%±2.3%.

[0060] 1.2 Modeling Drugs

[0061] Preparation of GBR12909 working solution: Take 10 mg of GBR12909 powder and prepare a mixed solvent according to the volume ratio of 2% DMSO, 40% PEG300, 5% Tween-80, and 53% physiological saline, and bring the volume to 10 mL. First, dissolve GBR12909 in DMSO, then add PEG300, Tween-80, and physiological saline sequentially, mixing constantly until completely dissolved, to obtain a clear working solution with a final concentration of 1 mg / mL. This solution should be freshly prepared and administered intraperitoneally within 1 hour of preparation; avoid storage.

[0062] High-fat diet: a diet in which fat provides energy at a rate of 60% or more.

[0063] 1.3 Modeling Methods and Grouping

[0064] After 7 days of acclimatization, mice were randomly divided into three groups: a control group (n=15), a GBR12909-only intervention group (n=15), and a phlegm-fire disturbance syndrome model group (n=15). The control group was fed standard diet and normal drinking water for 4 weeks, and then injected intraperitoneally with an equal volume of solvent at the same time every day for the following 14 days. The GBR12909-only intervention group was also fed standard diet and normal drinking water for 4 weeks, and then injected intraperitoneally with GBR12909 working solution (10 mg / kg) at the same time every day for the following 14 days. The phlegm-fire disturbance syndrome model group was fed a high-fat diet combined with 20% sucrose in drinking water for 4 weeks, and then injected intraperitoneally with the same dose of GBR12909 working solution (10 mg / kg) at the same time every day for the following 14 days.

[0065] Example 2

[0066] Application and verification of objective evaluation method for phlegm-fire disturbing the mind syndrome

[0067] 1.1 Evaluation of the TCM syndrome of phlegm-fire disturbing the mind

[0068] After a 4-week high-fat, high-sugar diet pretreatment followed by 14 days of continuous intraperitoneal injection intervention, mice in each group were evaluated for TCM phlegm-fire disturbance syndrome. The TCM phlegm-fire disturbance syndrome evaluation indicators included changes in hair luster, oral and nasal mucosa color, fecal characteristics, and water intake.

[0069] According to the scoring criteria shown in Table 1, the four indicators were initially scored from 1 to 5 points, where 1 point indicates the corresponding indicator is in a normal state, 2 to 4 points indicate progressively increasing abnormality, and 5 points indicate the highest degree of abnormality. The initial scores of the four indicators were added together to obtain the total initial score for the syndrome, which was then converted into a TCM phlegm-fire disturbing the mind syndrome score from 0 to 100 points. The calculation formula is as follows:

[0070]

[0071] The results showed that, compared with the control group, the mice in the phlegm-fire disturbing the mind syndrome group had significantly higher scores in hair luster, characterized by dull, slightly disheveled, or noticeably disheveled hair (P < 0.01); significantly higher scores in oral and nasal mucosal color, characterized by reddish or erythematous mucosa (P < 0.01); significantly higher scores in fecal characteristics, characterized by softer, loose, or sticky feces (P < 0.01); and significantly higher scores in water intake, indicating a significant increase in water consumption (P < 0.01). The total score of the phlegm-fire disturbing the mind syndrome in Traditional Chinese Medicine was significantly higher than that in the control group (P < 0.01). Figure 1 Compared with the control group, the GBR12909-only intervention group did not show significant increases in hair luster, oral and nasal mucosal color, fecal characteristics, or the total score of TCM phlegm-fire disturbance syndrome. Only the water intake score showed a slight increase (P < 0.05), suggesting that although GBR12909-only intervention can induce a certain increase in water intake related to the state of excitement, it is insufficient to stably induce the external manifestations related to phlegm-dampness-heat constitution. High-fat and high-sugar dietary intervention can induce more obvious external heat signs and phlegm-dampness-related manifestations in mice.

[0072] 1.2 Behavioral Dimension Evaluation

[0073] Twenty-four hours after the last GBR12909 injection, the behavioral testing phase begins. Following the predetermined behavioral testing protocol, the following tests are conducted sequentially: sucrose preference test, central open field test, forced swimming test, three-room social interaction test, rotarod test, elevated cross maze test, and water maze test. There is a minimum interval of 120 minutes between different tests conducted on the same day; the water maze test, involving continuous training, follows the procedure of training on days 8-12 and spatial exploration test on day 13.

[0074] 1.2.1 Sugar Water Preference Experiment

[0075] The experimental procedure was divided into an adaptation phase and a testing phase. The adaptation phase lasted for 2 days, with mice housed in individual cages. Each cage was provided with one bottle of clean water and one bottle of 2% sucrose solution. The positions of the two bottles were exchanged at 10:00 and 20:00 each day to eliminate positional bias. The testing phase began at 20:00 on the 4th day. The intake of clean water and sucrose solution for each group of mice was recorded over 12 hours. The sucrose preference index was calculated using the formula SPT = sucrose solution intake / total water intake × 100% to evaluate the mice's pleasure state.

[0076] The results showed that there was no significant difference in the sugar water preference rate between the GBR12909 intervention group and the phlegm-fire disturbance syndrome group and the control group. The overall sugar water preference rate of the three groups remained at a high level. Figure 2 The results indicate that neither GBR12909 intervention alone nor high-fat, high-sugar combined with GBR12909 intervention induced significant anhedonia, suggesting that the model animals did not exhibit typical depressive-like behaviors.

[0077] 1.2.2 Central Open Field Experiment

[0078] The central open field experiment was conducted at 10:00 AM on the 5th day after model establishment. The VisuTrack behavioral analysis system was used to record the movement trajectory of the mice in the open field device over 5 minutes, and the total movement distance and number of squares crossed were analyzed.

[0079] The total distance traveled by mice in the central open field experiment was used as the sole scoring indicator for the behavioral dimension. The mean total distance traveled by mice in the normal control group of the same evaluation batch was used as the baseline and set to 0 points. The maximum observed total distance traveled by mice in the phlegm-fire disturbance syndrome model group of the same evaluation batch was used as the upper limit and set to 100 points. The behavioral dimension scores for each mouse under evaluation were calculated according to the following formula:

[0080]

[0081] The results showed that, compared with the control group, the total movement distance and number of squares crossed in the GBR12909-only intervention group were significantly increased, suggesting that GBR12909 can induce increased spontaneous activity and exploratory behavior in mice; the total movement distance and number of squares crossed in the phlegm-fire disturbance syndrome group were also significantly higher than those in the control group, and significantly higher than those in the GBR12909-only intervention group, showing more obvious hyperactivity and enhanced exploratory behavior. Figure 3The above results indicate that GBR12909 intervention alone can induce a certain degree of increased manic-like activity. Furthermore, the combination of a high-fat, high-sugar diet pretreatment and GBR12909 further enhanced the hyperactivity in mice, suggesting that a constitution with phlegm-dampness and heat accumulation may amplify the manic-like behavioral response induced by dopaminergic stimulation. The total distance traveled in the central open field test was included as a core scoring indicator for the behavioral dimension in the total score calculation, and the number of grid crossings was used to support the determination of manic-like behavioral characteristics in the model.

[0082] 1.2.3 Forced Swimming Experiment

[0083] A preliminary experiment was conducted at 13:00 on day 5 after modeling, lasting 6 minutes; a formal experiment was conducted at 8:00 on day 6, and the immobility time of the mice within the last 4 minutes was recorded to evaluate the level of behavioral inhibition and the ability to maintain activity under stress.

[0084] The results showed that, compared with the control group, the immobility time of mice in the GBR12909-only intervention group was significantly reduced (P < 0.01), suggesting that GBR12909 alone intervention can reduce the level of behavioral inhibition in mice under stress. The immobility time of mice in the phlegm-fire disturbance syndrome group was also significantly reduced compared with the control group (P < 0.01), but there was no statistically significant difference compared with the GBR12909-only intervention group (P > 0.05) (Figure 4). These experimental results were used to assist in the analysis of the model's behavioral characteristics and were not included in the total score of the behavioral dimensions.

[0085] 1.2.4 Three-room social experiment

[0086] The three-compartment social experiment was conducted at 8:00 on day 7 after modeling to evaluate the social motivation and social novelty preference of the mice.

[0087] The results showed that, in terms of social interaction index, there was no significant difference between the GBR12909-only intervention group and the control group (P > 0.05), while the social interaction index of mice in the phlegm-fire disturbance syndrome group was significantly higher than that of the control group (P < 0.01), and significantly higher than that of the GBR12909-only intervention group (P < 0.01). Figure 5Regarding the novelty social preference index, there was no significant difference between the GBR12909-only intervention group and the control group (P > 0.05), while the novelty social preference index of the phlegm-fire disturbance syndrome group was significantly higher than that of the control group and the GBR12909-only intervention group (P < 0.01) (Figure 5). These results indicate that while GBR12909-only intervention can induce increased activity and excitability in mice, it is insufficient to stably induce a significant increase in social motivation and novelty social preference. However, after high-fat, high-sugar diet pretreatment combined with GBR12909, mice exhibited stronger social approach behavior and responsiveness to new social stimuli, suggesting a more pronounced tendency towards social hyperactivity and extraversion. This behavioral characteristic is highly consistent with the clinical manifestations of increased activity, excessive socialization, and enhanced response to external stimuli in a manic state. These experimental results are used to assist in the analysis of the model's behavioral characteristics and are not included in the total score of the behavioral dimensions.

[0088] 1.2.5 Rotating Rod Experiment

[0089] The rotarod test was performed at 13:00 on the 6th day after modeling to evaluate the mice’s motor coordination and balance function, and to exclude the interference of basic motor function abnormalities on other behavioral test results.

[0090] The results showed that there was no significant difference in the retention time on the rotarod between the GBR12909 single intervention group and the phlegm-fire disturbance syndrome group and the control group (P > 0.05); at the same time, there was also no significant difference between the phlegm-fire disturbance syndrome group and the GBR12909 single intervention group (P > 0.05). Figure 6 The results indicate that neither GBR12909 intervention alone nor the combined high-fat, high-glucose intervention with GBR12909 caused damage to the basic motor coordination and balance function of mice. This suggests that the behavioral changes observed in the central open field test, such as increased total distance traveled and number of squares crossed, and decreased immobility time in the forced swimming test, are not due to motor dysfunction, abnormal balance, or decreased exercise tolerance, but rather primarily reflect manic-like behavioral characteristics such as increased spontaneous activity, heightened excitability, and decreased behavioral inhibition in the model mice. These experimental results are used to assist in the analysis of the model's behavioral characteristics and are not included in the total score of the behavioral dimensions.

[0091] 1.2.6 Elevated Cross Maze Experiment

[0092] The elevated cross maze test was conducted at 18:00 on the 5th day after the model was completed to help distinguish between manic-like behavior and anxiety-like behavior.

[0093] The results showed that there was no significant difference in the open arm preference index between the GBR12909 single intervention group and the phlegm-fire disturbance syndrome group and the control group (P > 0.05); at the same time, there was also no significant difference between the phlegm-fire disturbance syndrome group and the GBR12909 single intervention group (P > 0.05). Figure 7 The results indicate that neither GBR12909 intervention alone nor high-fat, high-sugar combined with GBR12909 intervention induced significant anxiety-like behavioral abnormalities. These results further support the good specificity of the animal model constructed in this invention at the behavioral level, effectively distinguishing between manic-like and anxiety-like behaviors. These experimental results are used to assist in the analysis of the model's behavioral characteristics and are not included in the total score of the behavioral dimensions.

[0094] 1.2.7 Water Maze Experiment

[0095] Water maze training was conducted on days 8-12 after modeling, with 4 training sessions per day, and the changes in escape latency were recorded for each group of mice. On day 13 at 8:00, a spatial exploration experiment was conducted, and the time spent in the target quadrant and the number of times the original platform was crossed were recorded after the platform was removed.

[0096] The results showed that the escape latency of mice in all groups gradually shortened with increasing training days, indicating that all three groups of mice were able to gradually complete the spatial learning process. Among them, the escape latency of mice in the GBR12909-only intervention group and the phlegm-fire disturbance syndrome group decreased more significantly than that of the control group. The phlegm-fire disturbance syndrome group showed a shorter escape latency in the later stages of training, suggesting that it did not show any impairment in spatial learning ability after modeling and exhibited a certain trend of enhanced responsiveness. Regarding the time spent in the target quadrant, there was no significant difference between the GBR12909-only intervention group and the control group, nor between the phlegm-fire disturbance syndrome group and the control group. However, the time spent in the target quadrant in the phlegm-fire disturbance syndrome group was significantly longer than that in the GBR12909-only intervention group (P < 0.05). Regarding the number of times the original platform was crossed, there was no significant difference between the GBR12909-only intervention group and the control group. The number of times the original platform was crossed in the phlegm-fire disturbance syndrome group was significantly higher than that in the control group (P < 0.05), but the difference was not statistically significant compared with the GBR12909-only intervention group. Figure 8 ).

[0097] The above results indicate that neither GBR12909 intervention alone nor the combined high-fat, high-glucose intervention with GBR12909 caused significant impairment in spatial learning and memory. Specifically, the phlegm-fire disturbance syndrome group showed an increased tendency to stay in the target area and cross the original platform more frequently during the spatial exploration phase, suggesting that while exhibiting manic-like hyperactivity, enhanced exploration, and increased social responsiveness, there was no decline in cognitive function; instead, they displayed certain characteristics of heightened responsiveness and spatial memory retention. These experimental results were used to assist in analyzing the model's behavioral characteristics but were not included in the total score of the behavioral dimensions.

[0098] 1.3 Evaluation of metabolic indicators

[0099] 1.3.1 Evaluation Method

[0100] The body weight of mice in each group was measured before and at the end of modeling, and fasting blood glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol levels were measured. The animal weight gain rate was calculated using the following formula:

[0101]

[0102] Animal weight gain rate, fasting blood glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol were used as metabolic evaluation indicators, and each indicator was converted to a score of 0 to 100.

[0103] For animal weight gain rate, fasting blood glucose, triglycerides, total cholesterol, and low-density lipoprotein cholesterol, the mean value of the corresponding indicators in the normal control group mice of the same evaluation batch was used as the baseline value and set to 0 points, and the maximum observed value of the corresponding indicators in the model group mice of the same evaluation batch was used as the upper limit value and set to 100 points. The scores of the corresponding indicators of the mice to be evaluated were calculated according to the following formula:

[0104]

[0105] The baseline value is the mean value of the corresponding indicator of normal control mice in the same evaluation batch, and the upper limit value is the maximum observed value of the corresponding indicator of model mice in the same evaluation batch.

[0106] For high-density lipoprotein cholesterol (HDL-C), the mean HDL-C value of normal control mice in the same evaluation batch was used as the baseline and set to 0 points, and the minimum observed HDL-C value of model mice in the same evaluation batch was used as the lower limit and set to 100 points. The index score was calculated according to the following formula:

[0107]

[0108] The lower limit value is the minimum observed value of high-density lipoprotein cholesterol in mice of the same evaluation batch model group.

[0109] When the calculated score for any single indicator is less than 0, it is recorded as 0 points; when it is greater than 100 points, it is recorded as 100 points. When the upper limit of an elevated indicator is less than or equal to the corresponding benchmark value, or the lower limit of high-density lipoprotein cholesterol is greater than or equal to the corresponding benchmark value, the corresponding single indicator score is recorded as 0 points.

[0110] After each individual indicator is converted to a percentage, the metabolic indicator dimension score is calculated according to the preset weights:

[0111]

[0112] The scores for the metabolic indicators range from 0 to 100. The higher the score, the greater the degree of abnormality in glucose and lipid metabolism in the mice being evaluated.

[0113] 1.3.2 Evaluation Results

[0114] The results showed that fasting blood glucose, triglycerides, total cholesterol, low-density lipoprotein cholesterol, and weight gain rate were significantly higher in the phlegm-fire disturbing the mind group than in the control group, while high-density lipoprotein cholesterol was significantly lower, indicating that the model group had formed a stable state of glucose and lipid metabolism disorder.

[0115] 1.4 Inflammation-Neurotransmitter Evaluation:

[0116] 1.4.1 Evaluation Method

[0117] Serum interleukin-6, interleukin-1β, tumor necrosis factor-α, striatal dopamine levels, and the prefrontal cortex glutamate / γ-aminobutyric acid ratio were measured. For each inflammatory neurotransmitter evaluation index, the mean value of the corresponding index in the normal control group mice of the same evaluation batch was used as the baseline value and set as 0 points, and the maximum observed value of the corresponding index in the phlegm-fire disturbance syndrome model group mice of the same evaluation batch was used as the upper limit value and set as 100 points. The scores of each index of the mice to be evaluated were calculated according to the following formula:

[0118]

[0119] The baseline value is the mean value of the corresponding indicator in the normal control group mice of the same evaluation batch, and the upper limit value is the maximum observed value of the corresponding indicator in the phlegm-fire disturbance syndrome model group mice of the same evaluation batch.

[0120] When the calculated score of any single indicator is less than 0, it is recorded as 0 points; when it is greater than 100 points, it is recorded as 100 points. When the upper limit value is less than or equal to the benchmark value, the corresponding single indicator score is recorded as 0 points.

[0121] Serum interleukin-6, serum interleukin-1β, serum tumor necrosis factor-α, striatal dopamine levels, and the prefrontal cortex glutamate / γ-aminobutyric acid ratio each have a weight of 20% in the inflammation-neurotransmitter dimension. The inflammation-neurotransmitter dimension score is calculated according to the following formula:

[0122]

[0123] The inflammation-neurotransmitter dimension score ranges from 0 to 100. The higher the score, the more pronounced the inflammation activation and neurotransmitter imbalance in the mouse being evaluated.

[0124] 1.4.2 Evaluation Results

[0125] The results showed that the serum levels of interleukin-6, interleukin-1β, tumor necrosis factor-α, striatal dopamine, and the glutamate / γ-aminobutyric acid ratio in the prefrontal cortex of mice in the phlegm-fire disturbing the mind syndrome group were significantly abnormal compared with those in the control group, suggesting that the model group had a relatively clear state of systemic inflammatory activation and central neurotransmitter imbalance.

[0126] 1.5 Model Comprehensive Score Calculation and Evaluation Criteria

[0127] 1.5.1 Calculation method for total score of evaluation indicators

[0128] After converting the scores for the TCM phlegm-fire disturbing the mind syndrome dimension, metabolic index dimension, inflammation-neurotransmitter dimension, and behavioral dimension to a range of 0-100 points, the model's comprehensive score was calculated according to preset weights. The weights for the TCM phlegm-fire disturbing the mind syndrome dimension, metabolic index dimension, inflammation-neurotransmitter dimension, and behavioral dimension were 15%, 25%, 35%, and 25%, respectively. The model's comprehensive score was calculated using the following formula:

[0129]

[0130] The comprehensive score of the model ranges from 0 to 100 points. The higher the score, the greater the degree of comprehensive abnormality of the mouse under evaluation in terms of TCM syndrome, glucose and lipid metabolism, inflammation-neurotransmitters, and manic-like behavior.

[0131] 1.5.2 Evaluation Criteria

[0132] The animal model of manic-depressive disorder (MDD) is considered successfully established when the overall model score of the mouse being evaluated is greater than or equal to 60 points, and the behavioral dimension score is greater than or equal to 60 points. Specifically, an overall model score greater than or equal to 60 points indicates that the weighted average degree of abnormality in the four evaluation dimensions of the mouse being evaluated reaches the preset modeling level; a behavioral dimension score greater than or equal to 60 points indicates that the mouse being evaluated exhibits a relatively clear core behavioral phenotype of manic-like hyperactivity. If either the overall model score or the behavioral dimension score is less than 60 points, the mouse being evaluated is considered not to have met the criteria for successful animal model establishment.

[0133] Using a dual threshold of model-based comprehensive scoring and behavioral dimension scoring can avoid the situation where high scores in metabolic, inflammation, or syndrome dimensions mask deficiencies in the core behavioral phenotype.

[0134] 1.5.3 Evaluation Results

[0135] The mice were comprehensively evaluated using the aforementioned four-dimensional weighted scoring method. The results showed that the average percentage scores for the TCM syndrome, metabolism, inflammation-neurotransmitter, and behavioral dimensions in the phlegm-fire disturbing the mind syndrome group were 71.33, 79.92, 50.17, and 96.67, respectively. After four-dimensional weighting, the comprehensive scores of each mouse in the phlegm-fire disturbing the mind syndrome group ranged from 64.44 to 87.06, with an average comprehensive score of 72.40, all exceeding the 60-point threshold. Furthermore, the behavioral dimension scores of each mouse were all above 60 points. These results indicate that the phlegm-fire disturbing the mind syndrome manic animal model constructed in this invention is stable and exhibits both manic-like behavioral abnormalities and phlegm-fire disturbing the mind syndrome characteristics. The weighted scoring results for each mouse in the phlegm-fire disturbing the mind syndrome and GBR12909 groups are shown in Table 2.

[0136] Table 2. Four-dimensional scores and overall model scores of mice in the phlegm-fire disturbing the mind syndrome model group.

[0137]

[0138] In summary, the embodiments of this invention have the following beneficial effects: This invention innovatively combines the etiology and pathogenesis theory of phlegm-fire disturbing the mind syndrome in traditional Chinese medicine with modern neurobiology to construct an animal model of mania with clear TCM syndrome attributes, and establishes a corresponding objective evaluation method for phlegm-fire disturbing the mind mania. The core innovation of this model lies in the fact that the metabolic-inflammatory internal environment induced by a high-fat, high-sugar diet provides the pathological constitutional basis of "phlegm-dampness-heat accumulation" for the dopaminergic amplification effect of GBR12909. The two synergistically reproduce the complete pathological chain of "phlegm-fire disturbing the mind and causing mania" in traditional Chinese medicine, which is different from the existing single GBR12909 model. This model is standardized in operation, has a high model success rate, and has clear TCM syndrome attributes, which can provide an effective tool for basic research on the prevention and treatment of mania with traditional Chinese medicine.

[0139] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an animal model of manic-depressive disorder caused by phlegm-fire disturbing the mind, characterized in that, Four-week-old male C57BL / 6J mice were selected and fed a high-fat diet and 20% sucrose drinking water for four weeks to simulate the constitution of phlegm-dampness-heat accumulation. Starting from the fifth week, GBR12909 was injected intraperitoneally at the same time every day for 14 consecutive days to induce manic-like behavior. The high-fat feed described above has a fat energy content greater than or equal to 60%. The GBR12909 is prepared into a 1 mg / mL working solution using a solvent consisting of 2% DMSO, 40% PEG300, 5% Tween-80 and 53% physiological saline, with an intraperitoneal injection dose of 10 mg / kg. The same time point is intraperitoneal injection from 8:00 to 10:00 every day to ensure the consistency of the diurnal rhythm.

2. The evaluation method for the animal model of manic-depressive disorder prepared according to claim 1, characterized in that, Step 1: Evaluate the mice's behavior, TCM phlegm-fire disturbance syndrome, metabolism, and inflammation-neurotransmitter indicators, and obtain dimensional scores; Step 2: Calculate the comprehensive score according to the formula: Comprehensive score = TCM phlegm-fire disturbing the mind syndrome score × 15% + Metabolic index score × 25% + Inflammation-neurotransmitter score × 35% + Behavioral score × 25%. Step 3: When the overall model score is greater than or equal to 60 points and the behavioral dimension score is greater than or equal to 60 points, the animal model of phlegm-fire disturbing the mind and mania is determined to be successfully constructed.

3. The evaluation method according to claim 2, characterized in that, The objective evaluation method for the indicators mentioned in step one is as follows: The behavioral evaluation index is the total distance traveled in the central open field experiment. The evaluation indicators for the TCM syndrome of phlegm-fire disturbing the mind are hair luster, color of oral and nasal mucosa, stool characteristics and changes in water intake. The metabolic evaluation indicators are animal weight gain rate, fasting blood glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol; The inflammation-neurotransmitter evaluation indicators are serum IL-6, IL-1β, TNF-α, striatal dopamine levels, and the prefrontal cortex glutamate / γ-aminobutyric acid ratio.

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